Biotin-MUC1 Peptide (27-mer), GalNAc-Transferase Activity Assay

Description:

SBH biotin-MUC1 peptide is a good acceptor substrate for a variety of GalNAc-transferases, including GalNAc-T2, T3 & T5, with 5 potential sites for O-linked glycosylation. In conjunction with our SBH-Tn antibody, biotin-MUC1 peptide can be used as an assay for GalNAc-Transferase activity (please contact SBH Sciences for assay details). The SBH biotin-MUC1 peptide sequence is taken from the immunodominant region within the tandem repeat domain of Mucin 1 (MUC1). MUC1 is a large, transmembrane glycoprotein expressed by most glandular epithelial cells, and has been found to have an exceptionally high content of O-linked glycans (1). MUC1 is also an important tumor associated antigen, both over-expressed and aberrantly glycosylated in adenocarcinomas (2). Upon request we can also provide O-linked biotin-MUC1 glycopeptides, pre-modified with O-GalNac or a variety of extended O-glycans.

MUC1 --> TnMUC1 – Biotin-MUC1 (20ug/ml) is mixed with UDP-GalNAc (0.5mM) in activity assay buffer (25mM Tris pH 7.5, 150mM NaCl, 2.5mM MnCl2). The reaction mixture is split into two fractions, and to one fraction GalNAc-T2 (SBH-003-GalNAc-T2) is added at 10ug/ml. After overnight incubation at room temperature, the fraction with GalNAc-T2 has been converted to TnMUC1, demonstrating activity. The other fraction is used as the negative control (MUC1).

SBH-Tn ELISA –ELISAs were carried out by coating 96 well plates with avidin at 1 ug/ml, blocking and incubating with a 1:4 serial dilution of MUC1 or TnMUC1. After washing, plates were incubated with SBH-Tn monoclonal antibody. After washing, plates were incubated with a goat-anti-mouse (IgG+IgM)-HRP conjugate (Thermo 31446) at 1:1000 dilution. After washing, plates were developed with 2x ABTS (Southern Biotech 0202-01). Net OD was measured at 405 – 650 nm. As low as 30ng/ml TnMUC1 could be detected with the SBH-Tn antibody, with no detectable binding to MUC1 (see Figure 1 panel (B) above).

Usage: For research only. Not for use in diagnostic or therapeutic procedures.